Immunoassays, which take advantage of natural immunological reactions, have found wide-spread use as analytical techniques in clinical chemistry. Because of the specificity of the reactions, they are particularly advantageous in quantifying biological analytes that are present in very low concentration in biological fluids. Such analytes include, for example, antigens, antibodies, therapeutic drugs, narcotics, enzymes, hormones, proteins, etc.
The analyte, which is the target of the assay is referred to herein as the ligand, and the labeled analyte is referred to as the labeled ligand (including immunocompetent derivatives and analogs of such ligand). Compounds which specifically recognize the ligand and the labeled ligand and react to form complexes with them are referred to herein as receptors. The receptor and the ligand or labeled ligand form a conjugate pair. Any member of the pair can function as a receptor or a ligand.
In competitive binding immunoassays, a labeled ligand is placed in competition with unlabeled ligand for reaction with a fixed amount of the appropriate receptor. Unknown concentrations of the ligand can be determined from the measured signal of either the bound or unbound (i.e. free) labeled ligand. The reaction proceeds as follows: EQU ligand+labeled ligand+receptor&lt;=&gt;ligand-receptor+labeled ligand-receptor.
Immunoassay analytical elements are known. In general, such elements comprise receptors, such as antibodies for a ligand, immobilized in a particulate layer. In addition the element usually contains a reagent system that, through interaction with a bound or unbound species, results in a signal that can be correlated to the concentration of ligand in a sample. In use, the sample is manually combined with an enzyme labeled ligand and applied to the element. After a time, a solution containing a substrate for the labeled ligand is applied to the particulate layer. The reaction with the substrate is catalyzed by the enzyme label to form a reaction product that ultimately causes a signal color to develop. The reflection density of the color can be correlated to the concentration of the ligand in the sample. Similar signal development systems are known for other known conventional labels such as radioactive tags, chromophores, fluorophores, stable free radicals, and enzyme cofactors, inhibitors and allosteric effectors.
Multilayer immunoassay elements are thin film elements which use the above described immunoassay principles to measure analytes in serum samples. In these elements, the rate of color formation is inversely correlated to the amount of analyte present. Also, the rate of color formation is directly proportional to the activity of the drug-labeled enzyme bound to the immobilized antibody. For the immunoassays to maintain a stable calibration, none of the enzyme activity (measured rate) can be lost in any of the slides during the specified calibration period.
Frequently, immunoassay elements are supplied to customers in plastic "cartridges" containing 50 separate elements from which one element may be removed at a time as needed. The elements are stacked one on top of another so that the lower 49 elements in the cartridge all have their top surfaces covered by the element above. However, the top element in the stack has no such covering, and therefore the surface of that element is exposed to environmental factors to which the other 49 elements are not. For example, the top (or first) element is more exposed to air flow and light than the remainder of the elements when the cartridges are being handled during manufacturing or when the cartridges are in the element supplies of the clinical analyzers.
During storage, prior to use, the cartridges themselves are stored in sealed, foil-lined bags. However, the top element is still more exposed to the residual air and humidity inside the sealed bags than the other 49 elements.
It has been found that, when a common test fluid was reacted with the elements in a cartridge, the rate of color formation observed in the top (or first) element was always lower than the rate of color formation observed when the same test fluid was applied to elements below the top element in the same cartridge.